UPDATES
Nitrile as Activating Group in the Asymmetric Bioreduction
drich) were added for the derivatization of acids 2a and 2b
to the corresponding methyl esters for GC analysis. After
30 min of incubation at room temperature, Na2SO4 was
added and after filtration the conversion and ee of methyl
esters of 2a and 2b were determined on GC. Control experi-
ments without addition of enzymes were performed regular-
ly.
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General Procedure for Deuterium Labelling Studies
Tris-HCl buffer (50 mM, pH 7.5) was prepared using D2O.
To 800 mL of this solution, containing substrates (E)-1a
methyl ester or (E)-1b (10 mM), NADH (15 mM), an ali-
quot of NCR and OPR1, respectively, were added (final
protein concentration 200 mgmLÀ1). The mixture was shaken
for 24 h at 308C and 120 rpm. This set-up was prepared ten-
fold. The combined samples were extracted with CDCl3 (2ꢃ
5 mL); the solution containing substrate (E)-1b was acidi-
fied to pH 1 using 4M HCl prior to extraction. The phases
were separated, denatured protein was removed by filtration
and the organic solvent was evaporated. The residue was
1
subjected to H NMR measurements using the following pa-
rameters: 16 scans and two dummy scans (48,000 data
points); relaxation delay: 1 s; acquisition time 4 s; spectral
width 6200 Hz; pulse sequence: 908 pulse, then acquisition.
[16] G. Oberdorfer, K. Gruber, K. Faber, M. Hall, Synlett
Supporting Information
2012, 1857–1864.
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Faber, Angew. Chem. 2007, 119, 4008–4011; Angew.
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Syntheses of (E)-1a, (Z)-1a, (E)-1b, (Z)-1b, rac-2a, (R)-2b,
crude 5, unlabelled and deuterium-labelled NMR spectra,
analytical methods, optimization of the bioreduction and
molecular modelling studies are available in the Supporting
Information.
Acknowledgements
[20] A. Muller, B. Hauer, B. Rosche, Biotechnol. Bioeng.
Part of this work was performed within the DK ꢀMolecular
Enzymologyꢁ and financial support by the Austrian Science
Fund (FWF, projects W9 and P22722) is gratefully acknowl-
edged. Part of the research has been performed within project
CHEM21, funded by the IMI (agreement n8115360 within
FP7/2007-2013 and EFPIA). Brian Jones is thanked for his
help in obtaining HR-MS analyses and Peakdale Molecular
is thanked for assistance in preparation of materials.
2007, 98, 22–29.
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